The project has two experimental approaches, one investigating the bidirectional communication between parasympathetic nerve development and SMG epithelial morphogenesis, and the second identifying embryonic mouse salivary gland stem/progenitor cells. Over the past year we have made advances in understanding the bidirectional communication between parasympathetic nerve development and SMG epithelial morphogenesis. Both parasympathetic and sympathetic branches of the autonomic nervous system innervate the adult SMG, but it is the parasympathetic axons that extend from the parasympathetic submandibular ganglion (PSG) and innervate the developing SMG. We have determined that mechanical removal of the PSG in SMG organ culture reduces epithelial branching and secondary duct formation. In addition, chemical inhibition of acetylcholine (ACh) release, chemical antagonism of muscarinic receptor activity, and gene knockdown of the M1 muscarinic receptor in the epithelium all have a similar effect as removing the PSG. In gain of function studies, ACh analogues also increase epithelial branching. These data suggest that neuronal-epithelial communication via ACh signaling provides a mitogenic and morphogenic signal to the SMG epithelium. Our ongoing studies will identify how the M1 receptor signal influences FGF10 proliferative signals in the epithelium.[unreadable] Given that the PSG axons migrate in a unidirectional manner along ducts towards the end buds we hypothesized that a molecule secreted by these buds was guiding axon migration. Microarray analysis of intact SMGs at early stages of development identified upregulation of Neurturin (Nrtn), a neurotrophic growth factor, and its receptor GFRa2, as SMG branching began. We separated epithelium from the mesenchyme and used Agilent whole genome microarray analysis to compare the gene expression in both tissue compartments. Nrtn and its receptor GFRa2 were expressed in the epithelium and mesenchyme, respectively. Furthermore, the transmembrane receptor Ret, which forms the functional signaling complex with GFRa2, was also expressed predominately by the mesenchyme. Nrtn and GFRa2 were differentially expressed in the epithelial buds and mesenchyme, respectively; GFRa2 protein was localized to the parasympathetic ganglion and axons. We added function-blocking Nrtn antibodies to SMG organ culture, which not only decreased axon outgrowth but also reduced epithelial growth and branching during culture. Addition of recombinant Nrtn to the culture medium increased epithelial bud formation; however, axon bundles migrated aberrantly into the mesenchyme, suggesting the directional Nrtn gradient towards the end buds was disrupted. The aberrant Nrtn-mediated axon outgrowth into the mesenchyme was inhibited by chemical inhibitors of Nrtn/GFRa2/RET signaling. Our results suggest that epithelial-neuronal communication, via Nrtn/GFRa2/Ret signaling, regulates both neuronal and epithelial morphogenesis during SMG development. [unreadable] We recently began a new research project and are developing methods to dissociate embryonic SMGs into single cell suspension, while maintaining the cell surface expression of markers, and have begun FACS analysis to identify progenitor/stem cell subpopulations of cells within the embryonic SMG. We will also analyze the growth, behaviour, and development of these embryonic gland stem/progenitor cells in ex vivo culture system to define important environmental parameters for regeneration. We will use stem cell markers previously identified in adult salivary glands and compare their expression in the embryonic SMG.[unreadable] In conclusion, our studies will provide a rationale for a biologically-based therapeutic approach for regeneration of irradiated salivary gland tissue. Understanding the cellular processes involved in tissue morphogenesis is critical to regenerating tissue.